Vertebrates rely on innate immune receptors to sense conserved structures from invading microbes. The overall goal of this project is to understand at the molecular level how innate immune receptors recognize flaviviruses, and how this recognition is translated into an immune response. We focus on innate immune recognition of viral genomic RNA in the cytoplasm by the DExD/H-box RNA helicases RIG-I and MDA5. RIGI recognizes short RNA oligonucleotides bearing 5'-triphosphates, while MDA5 recognizes longer, double stranded RNAs. Our first goal is to measure the binding affinities of RIG-I and MDA5 for RNA substrates of various lengths and secondary structures. We will begin to dissect the mechanism of signal transduction of RIG-I and MDA5 by measuring their rates of ATP hydrolysis and their helicase activities in the presence of their preferred RNA substrates. In the major part of our proposal, we will determine the crystal structures of RIG-I and MDA5 in the presence and absence of their preferred RNA ligands. These crystal structures will reveal the conformational changes caused by RNA binding, which will allow us to understand how binding of RNA is translated into an innate immune signal. To test our structure-based model for signal generation, we will engineer mutations designed to alter the signaling and ligand-binding properties of RIG-I and MDA5. Intracellular signaling in response to viral genomic RNA is modulated by another DExD/H-box helicase, LGP2, which interacts directly with RIG-I and MDA5. To understand how LGP2 regulates RIG-I and MDA5 signaling, we will determine the crystal structure of LGP2 in complex with RIG-I or MDA5. Paramyxoviruses inhibit MDA5 signaling by binding MDA5 directly with their V proteins. In order to understand the molecular basis of this viral immune evasion, we will determine the crystal structure of MDA5 in complex with SV5 V. Our structural approach will provide unique mechanistic insights into the generation and regulation of viral RNA sensing by RIG-I and MDA5. In the long term, this work will guide the rational design of novel vaccine adjuvants and immunomodulatory therapeutics targeting RIG-I or MDA5, thus providing new strategies for the prevention and treatment of viral infections.